PROJECT SUMMARY Acute Myeloid Leukemia (AML) is a cancer characterized by a co-operative block in hematopoietic maturation and proliferative advantage. It is near incurable, with a 5-year survival rate of only 25% and median survival <1year in patients over 65 years. Current chemotherapy is very poorly tolerated and majority of patients relapse. Overcoming the differentiation block is a promising therapeutic avenue, however is majorly lacking for AML subtypes without a known driver oncogene. Our study examines new pathways to target the two major aspects of leukemic cell survival, the differentiation block and increased proliferative ability, that can prove effective across multiple subtypes. Reversible protein phosphorylation controls many aspects of such cell fate decisions, and an imbalance in the normal activities of kinases and phosphatases contribute to pathogenesis of AML. However, while the aberrant activation of kinases, frequent in AML, is well studied and exploited for therapeutic purposes, inactivation of phosphatases, also prevalent in AML, is often overlooked. This proposal focuses on the serine threonine phosphatase tumor suppressor Protein Phosphatase 2A (PP2A) and its role in influencing proliferation vs differentiation decisions in AML. For the F99 phase, we identified a novel role for PP2A in driving myeloid differentiation and cell cycle arrest in AML. Based on our preliminary data, our overall hypothesis is that PP2A drives differentiation and cell cycle arrest through the cell cycle regulator p21. We specifically seek to establish the mechanism of PP2A mediated p21 induction, and the role of potential downstream factors Retinoblastoma protein and CEBP proteins in driving differentiation in AML using genetic overexpression and knockdown systems, CHIP-Seq and single cell analysis using Mass Cytometry. The K00 phase focuses on identification of factors mediating DNA damage induced differentiation. While DNA damage has been shown to induce differentiation in AML and hematopoietic progenitors, the molecular mechanism is poorly understood. Interestingly, DNA repair machinery including ATM and ATR serine threonine kinases protect leukemic cells from damage induced differentiation and are hyperactivated in AML. This raises the possibility that inhibiting DNA repair can promote genomic stress induced differentiation. My hypothesis for the K00 phase is that PP2A can antagonize DNA repair kinases and promote differentiation in response to DNA damage in AML. I will establish the role of PP2A and other potential candidates identified from preliminary data and literature such as GADD45 proteins and p21 in DNA damage induced differentiation in AML using genetic and pharmacological activation of PP2A, overexpression, knockdown and knockout of GADD45 as well as global in-vitro and in-vivo RNAi and genome wide CRISPER screen analyses. Successful completion of this proposal will yield new insight into cellular pathways capable of inducing growth arrest and terminal differentiation of maturation blocked cancers such as AML.